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Aims: The diagnosis of thyroid neoplasms relies upon the demonstration of histological parameters that can be focal and prone to subjective interpretation. We evaluated the utility of NRAS Q61R immunohistochemistry (IHC) in the diagnosis of thyroid lesions after determining its specificity and sensitivity as a surrogate marker for RAS Q61R mutation., Method and Results: NRAS Q61R IHC was performed on 282 primary or metastatic thyroid lesions from 256 patients. RAS mutation status was collected from patients' charts. Sensitivity and specificity of NRAS Q61R IHC for detecting a RAS Q61R mutation was calculated. IHC-positive cases were reviewed to determine the diagnostic utility of NRAS Q61R IHC. NRAS Q61R immunopositivity was seen in non-neoplastic, benign and malignant thyroid lesions. NRAS Q61R antibody cross-reactivity led to the detection of NRAS Q61R, KRAS Q61R and HRAS Q61R proteins. Among primary thyroid carcinomas, immunopositivity was most frequent in papillary thyroid carcinomas, follicular variant (48.0%). The sensitivity and specificity of NRAS Q61R IHC in detecting RAS Q61R mutation was 90.6% and 92.3%, respectively. When positive, the NRAS Q61R stain was determined to be helpful in demonstrating infiltration, tumour size, capsular and/or vascular invasion and multifocality., Conclusion: NRAS Q61R IHC is highly sensitive and specific for the detection of RAS Q61R mutations in thyroid pathology and is particularly relevant in follicular-patterned neoplasms. When evaluated alongside histological features, NRAS Q61R immunoreactivity can be instrumental in the diagnosis and classification of thyroid nodules., (© 2021 John Wiley & Sons Ltd.)

IDH2 R172 mutations occur in >80% sinonasal undifferentiated carcinomas ("SNUC") and ~80% of these are R172S and R172T variants. We examined the utility of the monoclonal antibody 11C8B1 to IDH2 R172S in IDH2 R172-mutated tumors to establish an immunohistochemistry protocol as a surrogate method for IDH2 R172S mutation detection. Eighty-eight formalin-fixed paraffin-embedded tumors including 42 sinonasal tumors and a variety of IDH1/2-mutated malignancies were tested by immunohistochemistry. The IDH1/2 mutation status was determined in 86 cases by a targeted massively parallel sequencing MSK-IMPACT TM assay. Interestingly, monoclonal antibody 11C8B1 was reactive with all IDH2 R172S (N = 15) mutated tumors including 12 sinonasal carcinomas, 2 high-grade sarcomas and one intrahepatic cholangiocarcinoma, and with all R172T (N = 3) mutated sinonasal carcinomas displaying a distinct granular cytoplasmic labeling in all R172S/T mutated malignancies. 11C8B1 immunohistochemistry was also positive in 2 of 6 IDH1 R132S-mutated tumors, including one intrahepatic cholangiocarcinoma and one chondrosarcoma showing a smooth homogeneous cytoplasmic staining pattern. All IDH2 R172G/K/M/W (N = 22) and IDH1 132H/C/G/L (N = 15) mutated tumors, and all IDH1/2-wild-type tumors (N = 25), including a histologic variety of 23 sinonasal tumors, were immunonegative. Importantly, 11 sinonasal undifferentiated carcinomas (N = 14, 79%) and 3 (100%) high-grade neuroendocrine carcinomas, large cell type were 11C8B1 immunopositive. Literature search revealed a virtual absence of IDH2 R172 and IDH1 R132S mutations in >1000 cases of 8 different malignancies included in the differential diagnosis of sinonasal undifferentiated carcinoma. Our study suggests that positive IDH2 11C8B1 immunohistochemistry in sinonasal carcinomas would be highly predictive of the presence of IDH2 R172S/T mutations and could serve as a reliable adjunct diagnostic marker of sinonasal undifferentiated carcinomas in >70% cases.

ETV6-NTRK3 fusion was identified in several cancers including the recently described mammary analog secretory carcinoma (MASC) of the salivary glands and a minority of papillary thyroid carcinomas. We describe three cases of primary MASC of the thyroid gland and provide a detailed clinical and pathological characterization of the tumor morphology, immunoprofile, and genetic background. Immunohistochemistry for PAX8, TTF-1, thyroglobulin, mammaglobin, GCDFP-15, S-100 protein, and p63 was used to define the tumor immunophenotype. Fluorescence in situ hybridization for ETV6 rearrangement was performed in three, and the next-generation sequencing assay MSK-IMPACT™ (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets) was performed in two cases. Primary MASC of the thyroid occurred in two women and one man, age 47-72 years. All patients presented with high T stage, infiltrative, locally aggressive tumors with extrathyroidal extension. Two cases were associated with well-differentiated papillary thyroid carcinoma. Histologically, they appeared as low-grade tumors, resembling MASC of the salivary glands and labeled positive for mammaglobin, GCDFP-15, S-100 protein, p63, weakly positive for PAX8, and negative for TTF-1 and thyroglobulin. Fluorescence in situ hybridization revealed ETV6 rearrangement in all cases. In two tested cases MSK-IMPACT™ confirmed the presence of ETV6-NTRK3 gene fusion. Two patients had at least two local recurrences, one was alive with disease, and one was alive and free of disease after 14 and 17 years, respectively. The third patient was alive and free of disease after 2 years. MASC of the thyroid is histologically, immunophenotypically, and genetically similar to its salivary gland counterpart. Thyroid MASC can be associated with a well-differentiated papillary thyroid carcinoma component, supporting follicular cell origin. Clinically, these carcinomas may show frequent recurrences but are associated with long-term survival. Patients with MASC of the thyroid may potentially benefit from Trk molecular-targeted therapy., Competing Interests: Disclosure/conflict of interest The authors declare no conflict of interest.

Sarcomas arising in the sinonasal region are uncommon and encompass a wide variety of tumor types, including the newly described biphenotypic sinonasal sarcoma (BSNS), which is characterized by a monomorphic spindle cell proliferation with dual neural and myogenic phenotypes. Most BSNSs harbor a pathognomonic PAX3-MAML3 fusion driven by t(2;4)(q35;q31.1), whereas the alternative fusion partner gene remains unidentified in a subset of PAX3-rearranged cases. As NCOA1 on 2p23 is a known partner in PAX3-related fusions in other tumor types (ie, alveolar rhabdomyosarcoma), we investigated its status by fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction assays in 2 BSNS cases showing only PAX3 gene rearrangements. Novel PAX3-NCOA1 fusions were identified in these 2 index cases showing an inv(2)(q35p23) by FISH and confirmed by reverse transcription polymerase chain reaction. Five additional BSNS cases with typical morphology were studied by FISH, revealing a PAX3-MAML3 fusion in 4 cases and only PAX3 rearrangement in the remaining case without abnormalities in MAML3 or NCOA1 gene. Except for 1 case with surface ulceration, all other tumors lacked increased mitotic activity or necrosis, and all cases immunohistochemically coexpressed S100 protein and actin, but lacked SOX10 reactivity. Interestingly, the 2 PAX3-NCOA1-positive cases showed desmin reactivity and displayed a small component of rhabdomyoblastic cells, which were not seen in the more common PAX3-MAML3 fusion cases. In conclusion, we report a novel PAX3-NCOA1 fusion in BSNS, which appears to be associated with focal rhabdomyoblastic differentiation and should be distinguished from PAX3-NCOA1-positive alveolar rhabdomyosarcoma or malignant Triton tumor. SOX10 immunohistochemistry is a useful marker in distinguishing BSNS from peripheral nerve sheath tumors.

Background: Overexpression of squamous cell carcinoma-related oncogene (SCCRO) is associated with invasive progression and poor outcomes in non-small cell lung cancer. We assessed the role of SCCRO as a tumor marker in bronchioloalveolar carcinoma (BAC), a subtype of adenocarcinoma exhibiting evidence of histologic tumor progression. We hypothesized that SCCRO expression would correlate with invasive tumor phenotypes and worse survival in BAC., Methods: We classified 150 tumors as pure BAC, BAC with focal invasion, or adenocarcinoma with BAC features. A tissue microarray was constructed from areas of benign lung, BAC, and invasive adenocarcinoma in these tumors. Squamous cell carcinoma-related oncogene expression was graded by immunohistochemistry from 0 to 3 (absent, low, moderate, or high), with positive SCCRO phenotype defined as grade 3. Squamous cell carcinoma-related oncogene specificity was determined by Wilcoxon rank test and area under the receiver-operator curve, survival by the Kaplan-Meier method, and correlation with prognostic factors by log-rank test., Results: Of the 86.0% (129 of 150) of specimens suitable for analysis, positive SCCRO phenotype was seen in 16.3% (21 of 129) and was 100.0% specific for tumor versus benign tissue (area under receiver-operator curve, 0.92). Positive SCCRO phenotype was greater in tumors with increasing degrees of invasive histologic type (7.0% pure BAC, 13.6% BAC with focal invasion, and 28.6% adenocarcinoma with BAC features; p = 0.02). Low-level SCCRO expression was present in 83.9% (99 of 118) of benign tissues and correlated with tobacco use and poor survival (p = 0.05)., Conclusions: Squamous cell carcinoma-related oncogene is a marker of invasive tumor progression in BAC. Low-level expression in adjacent benign lung predicts worse survival, and may represent field cancerization or host-tumor effects.

We compared histomorphological features and molecular expression profiles of adrenocortical adenomas (ACAd) and carcinomas (ACCa). A critical histopathological review (mean, 11 slides per patient) was conducted of 37 ACAd and 67 ACCa. Paraffin-embedded tissue cores of ACAd (n = 33) and ACCa (n = 38) were arrayed in triplicate on tissue microarrays. Expression profiles of p53, mdm-2, p21, Bcl-2, cyclin D1, p27, and Ki-67 were investigated by immunohistochemistry and correlated with histopathology and patient outcome using standard statistical methodology. Median follow-up period was 5 years. Tumor necrosis, atypical mitoses, and >1 mitosis per 50 high-power fields were factors that were highly specific for ACCa (P <.001). Number (0 to 4) of unfavorable markers [Ki-67 (+), p21 (+), p27 (+), mdm-2(-)] expressed was significantly associated with mitotic activity and morphologic index (i.e., number of adverse morphologic features) and highly predictive of malignancy (P <.001). Ki-67 overexpression occurred in 0 ACAd and 36% ACCa (P <.001) and was significantly associated with mitotic rate and unfavorable morphologic index (P <.001). Tumor necrosis, atypical mitoses, >5 mitoses per 50 high-power fields, sinusoidal invasion, histologic index of >5, and presence of more than two unfavorable molecular markers were associated significantly with metastasis in ACCa. Well-established histopathologic criteria and Ki-67 can specifically distinguish ACCAd from ACCa. Tumor cell proliferation (Ki-67) correlates with mitotic activity and morphologic index. Tumor morphology is a better predictor of metastatic risk in ACCa than current immunohistochemistry-detected cell cycle regulatory and proliferation-associated proteins.

The present study was performed to detect circulating prostatic carcinoma (PC) cells using a novel three-step immunobead reverse transcriptase (RT) polymerase chain reaction (PCR) assay for prostatic specific membrane antigen (PSMA) messenger RNA (mRNA). The sensitivity and specificity of this technique was assessed and the incidence of immunobead RT-PCR positivity correlated with progressive metastatic disease and serum prostatic specific antigen (PSA) levels. Fifty peripheral blood (PB) samples from 46 patients with PC were incubated with magnetic beads coated with Ber-EP4 antibody directed against the human epithelial antigen a membrane antigen widely expressed by epithelial cells. The epithelial cell-enriched magnetic fraction was then subjected to mRNA isolation using oligo-deoxythymidine (dT) magnetic beads. Nested RT-PCR for PSMA was performed on the mRNA oligo-dT complex and the identity of the RT-PCR products was confirmed by Southern blotting. Twenty-one PB samples from 8 control subjects without PC were also evaluated. Three-step immunobead PSMA RT-PCR was able to detect one PC cell per 1 mL of PB. The positivity rate of the RT-PCR assay was significantly higher (11 of 25; 44%) in patients with metastatic tumor than in patients with non-metastatic disease (1 of 21; 5%) (P = 0.003). In patients with metastatic PC, RT-PCR positivity was much higher in patients with progressive disease (10 of 13; 77%) than in patients with responding or stable disease (1 of 12; 8%) (P = 0.001). There was a statistically significant correlation between immunobead PSMA PCR positivity and high levels of serum PSA (P = 0.005). All control subjects without PC tested negative for PSMA PCR. The three-step immunobead RT-PCR for PSMA can detect circulating PC cells with high specificity and sensitivity. Preliminary data show a strong correlation between immunobead PCR positivity, the presence of progressive metastatic disease, and high levels of serum PSA.